How cellular proteins control cancer spread

New finding may help focus the search for anti-cancer drugs

A new insight into cell signals that control cancer growth and migration could help in the search for effective anti-cancer drugs. A team of researchers has revealed key biochemical processes that advance our understanding of colorectal cancer, the third most common cancer among Canadians.

Using the CMCF beamline at the Canadian Light Source (CLS) at the University of Saskatchewan, scientists from McGill University and Osaka University in Japan were able to unlock the behavior of an enzyme involved in the spread of cancer cells. The team found that there is a delicate interaction between the enzyme, PRL3, and another protein that moves magnesium in and out of cells. This interaction is crucial to colorectal cancer growth.

A new insight into cell signals that control cancer growth and migration could help in the search for effective anti-cancer drugs. A team of researchers has revealed key biochemical processes that advance our understanding of colorectal cancer, the third most common cancer among Canadians.

Using the CMCF beamline at the Canadian Light Source (CLS) at the University of Saskatchewan, scientists from McGill University and Osaka University in Japan were able to unlock the behavior of an enzyme involved in the spread of cancer cells. The team found that there is a delicate interaction between the enzyme, PRL3, and another protein that moves magnesium in and out of cells. This interaction is crucial to colorectal cancer growth.

Read more on the Canadian Light Source website

Image: Members of the Gehring research laboratory discussing the results of a protein purification.

Cross-β Structure – a Core Building Block for Streptococcus mutans Functional Amyloids

Most amyloids1 are misfolded proteins, having enormous variety in native structures. Pathological amyloids are implicated in diseases including Alzheimer’s disease and many others.  They are characterized by long, unbranched fibrillar structure, enhanced birefringence on binding Congo red dye, and cross-β structure – β-strands running approximately perpendicular to the fibril axis, forming long β-sheets running in the direction of the axis.  Fiber diffraction patterns from amyloids are marked by strong intensity at about 4.8 Å in the meridional direction (parallel to the fibril axis), corresponding to the separation of strands in a β-sheet, and in many cases broader but distinct equatorial intensity at about 10 Å.  The 10 Å intensity (whose position may vary considerably) comes from the distance between stacked β-sheets.  This stacking is characteristic of the many amyloids formed by small peptides, including peptide fragments of larger amyloidogenic proteins.  While some authors have required the 10 Å intensity to characterize an amyloid, it is not strictly necessary, since architecturally more complex examples have been found of Congo-red-staining fibrils with cross-β structure, but without the stacked-sheet structure, and consequently without the 10 Å intensity on the equator.

Amyloids do not always stem from protein misfolding.  Organisms across all kingdoms utilize functional amyloids in numerous biological processes.  Bacteria are no exception. Bacterial amyloids contribute to biofilm formation and stability.  Tooth decay is the most common infectious disease in the world.  A major etiologic agent, Streptococcus mutans, is a quintessential biofilm dweller that produces at least three different amyloid-forming proteins, adhesins P1 and WapP, and the cell density and competence regulator Smu_63c2.  The naturally occurring truncation derivatives of P1 and WapA, C123 and AgA, represent the amyloidogenic moieties, and a new paradigm of Gram-positive bacterial adhesins is emerging of adhesins having dual functions in monomeric and amyloid forms. While each S. mutans protein possesses considerable β-sheet structure, the tertiary structures of each protein are quite different (Fig. 1).  This study further characterized S. mutans amyloids and addressed the ongoing debate regarding the underlying structure and assembly of bacterial amyloids including speculation that they are structurally dissimilar from better-characterized amyloids.

Read more on the SLAC website

Image: Crystal or predicted 3D structures of S. mutans C123 (left), AgA (center), and Smu_63c (right).

Unravelling the secrets of the malaria parasite

PETRA III helps to identify a new kind of protein in Plasmodium falciparum

For the first time, scientists have identified a lipocalin protein in the malaria parasite Plasmodium falciparum. The discovery helps to better understand the life cycle of the parasite that is a major health burden in large parts of the world. The cooperation between the groups of Tim Gilberger from the Centre for Structural Systems Biology CSSB (Cellular Parasitology Department at Bernhard Nocht Institute for Tropical Medicine/ Universität Hamburg) at DESY and Matthias Wilmanns from the Hamburg branch of the European Molecular Biology Laboratory EMBL describes the discovery in the journal Cell Reports. CSSB is a cooperation of nine institutions, including DESY, that have deputed scientists to the centre.

With an estimated 228 million cases per year worldwide and more than 400,000 deaths, malaria remains one of the most important human health threats. There is no vaccine commercially available. While biologists have revealed many details about how the malaria parasite rapidly feeds on and transforms its host’s red blood cells, there are many unsolved mysteries surrounding the parasite’s life cycle. Using the microscopic facilities available at CSSB in combination with EMBL’s X-ray beamlines at DESY’s research light source PETRA III, the team unraveled a small piece of this mystery with the identification and characterization of the first lipocalin in the most virulent malaria parasite species P. falciparum.

Read more on the PETRA III (at DESY) website

Image: Ribbon diagram of the protein structure of Plasmodium falciparum Lipocalin PfLCN that comes in tertramers, i.e. complexes of four identical molecules. Fluorescence micrographs of the parasite (upper right and lower left) show that the lipocalin accumulates in vacuoles.

Credit: BNITM/EMBL, Paul-Christian Burda/Thomas Crosskey [Source]

Protecting chickens from heart disease

The health and welfare of broiler chickens may improve thanks to University of Saskatchewan (USask) researcher Andrew Olkowski and colleagues.

More chickens are raised worldwide than any other livestock animal, so improving their health outcomes would have a big impact.

The broiler chickens that are raised for meat were genetically selected to grow extremely fast, but they often suffer from heart diseases. Heart pump failure is a major health and welfare issue for the broiler chicken industry worldwide. Globally, economic losses associated with heart failure problems in broiler chickens amount to more than $1 billion annually.  

To understand why fast-growing broiler chickens suffer from heart problems, Olkowski and collaborators compared them with their slower-growing broiler counterparts, which have a much lower risk of heart failure, and with Leghorn chickens, which are resistant to heart failure.

Read more on the Canadian Light Source website

Image: University of Saskatchewan researcher Andrew Olkowski. 

Cell membrane proteins imaged in 3-D

Scientists used lanthanide-binding tags to image proteins at the level of a cell membrane, opening new doors for studies on health and medicine.

A team of scientists including researchers at the National Synchrotron Light Source II (NSLS-II)—a U.S. Department of Energy (DOE) Office of Science User Facility at DOE’s Brookhaven National Laboratory—have demonstrated a new technique for imaging proteins in 3-D with nanoscale resolution. Their work, published in the Journal of the American Chemical Society, enables researchers to identify the precise location of proteins within individual cells, reaching the resolution of the cell membrane and the smallest subcellular organelles.
“In the structural biology world, scientists use techniques like x-ray crystallography and cryo-electron microscopy to learn about the precise structure of proteins and infer their functions, but we don’t learn where they function in a cell,” said corresponding author and NSLS-II scientist Lisa Miller. “If you’re studying a particular disease, you need to know if a protein is functioning in the wrong place or not at all.”
The new technique developed by Miller and her colleagues is similar in style to traditional methods of fluorescence microscopy in biology, in which a molecule called green fluorescent protein (GFP) can be attached to other proteins to reveal their location. When GFP is exposed to UV or visible light, it fluoresces a bright green color, illuminating an otherwise “invisible” protein in the cell.

>Read more on the National Synchrotron Light Source II (NSLS-II) website

Image: Ultrabright x-rays revealed the concentration of erbium (yellow) and zinc (red) in a single E.coli cell expressing a lanthanide-binding tag and incubated with erbium.

Researchers use CHESS to map protein motion

Cornell structural biologists took a new approach to using a classic method of X-ray analysis to capture something the conventional method had never accounted for: the collective motion of proteins.

And they did so by creating software to painstakingly stitch together the scraps of data that are usually disregarded in the process.
Cornell structural biologists took a new approach to using a classic method of X-ray analysis to capture something the conventional method had never accounted for: the collective motion of proteins. And they did so by creating software to painstakingly stitch together the scraps of data that are usually disregarded in the process.
Their paper, “Diffuse X-ray Scattering from Correlated Motions in a Protein Crystal,”published March 9 in Nature Communications.
As a structural biologist, Nozomi Ando, M.S. ’04, Ph.D. ’08, assistant professor of chemistry and chemical biology, is interested in charting the motion of proteins, and their internal parts, to better understand protein function. This type of movement is well known but has been difficult to document because the standard technique for imaging proteins is X-ray crystallography, which produces essentially static snapshots.

>Read more on the CHESS website
>Read also: Diffuse X-ray Scattering from Correlated Motions in a Protein Crystal

Image: This slice through the three-dimensional diffuse map shows intense peaks resulting from lattice vibration, as well as cloudy features caused by internal protein motions.

ALS reveals vulnerability in cancer-causing protein

A promising anticancer drug, AMG 510, was developed by Amgen with the help of novel structural insights gained from protein structures solved at the Advanced Light Source (ALS).

Mutations in a signaling protein, KRAS, are known to drive many human cancers. One specific KRAS mutation, KRAS(G12C), accounts for approximately 13% of non-small cell lung cancers, 3% to 5% of colorectal cancers, and 1% to 2% of numerous other solid tumors. Approximately 30,000 patients are diagnosed each year in the United States with KRAS(G12C)-driven cancers.

Despite their cancer-triggering significance, KRAS proteins have for decades resisted attempts to target their activity, leading many to regard these proteins as “undruggable.” Recently, however, a team led by researchers from Amgen identified a small molecule capable of inhibiting the activity of KRAS(G12C) and driving anti-tumor immunity. Protein crystallography studies at the ALS provided crucial information about the structural interactions between the potential drug molecule and KRAS(G12C).

>Read more on the Advanced Light Source website

Image: A structural map of KRAS(G12C), showing the AMG 510 molecule in the binding pocket. The yellow region depicts where AMG 510 covalently attaches to the KRAS protein.
Credit: Amgen

Discovering a whole new family of copper-binding proteins

While studying a class of copper-containing enzymes, a team of researchers discovered and characterised a new family of fungal proteins.

Their study has now been published on Nature Chemical Biology, including analysis performed at BioMAX. The article is published together with a parallel study that sheds light on one of the potential biological roles of the proteins in this new family.

In contrast with a certain romanticised idea of research, scientific discoveries seldom come with a shouted “eureka!” as to mark the end of a linear intellectual endeavour. Much more frequently, new scientific findings emerge from observations where a scientist’s first reaction might sound like “that’s odd…”. Perhaps that was how the authors of this study reacted when they realised what they were looking at wasn’t what they were looking for.

In an article published this week on Nature Chemical Biology, a team of scientists from INRA, University of Copenhagen, Marseille Université, and University of York characterised a new family of proteins, named X325, found in various fungal lineages. The article is published together with a parallel study in which one protein of this new family, Bim1, is described as involved in fungal meningitis.

The authors were initially searching for new lytic polysaccharide monooxygenases (LPMOs), copper-dependent enzymes specialised in the degradation of polysaccharides and widely used in the production of biofuels. The proteins of this new family seemed promising candidates since they share many structural features and a probable common ancestor with LPMOs. However, the researchers proved that the members of this LPMO-like protein family are not involved in polysaccharides degradation, but they more likely play a role in the regulation of copper ion content in the organisms where they are expressed.

>Read more on the MAX IV website

Image: Copper binding site of two different proteins. Left: LaX325 protein belonging to the newly identified LPMO-like protein family X325. Right: cellulose cleaving LPMO enzyme TaAA9.
Image developed by Tobias Tandrup, University of Copenhagen.

Using European XFEL to shed light on photosynthesis

First membrane protein studied at European XFEL

In a paper now published in Nature Communications an international group of scientists show that the fast X-ray pulse rate produced by the European XFEL can be used to study the structure of membrane proteins such as those involved in the process of photosynthesis. These results open up eagerly awaited experimental opportunities for scientists studying these types of proteins.

Large proteins and protein complexes are difficult to study with traditional structural biology approaches. Large protein complexes, such as those that sit across cell membranes and regulate traffic in and out of cells, are difficult to crystalize and generally only produce small crystals that are hard to analyse. The extremely fast X-ray pulses generated by European XFEL now enable scientists to collect large amounts of data from a stream of small crystals to develop detailed models of the 3D structure of these proteins.

>Read more on the European XFEL website

Image (extract, full illustration in the article): Graphic shows the basic design of a serial femtosecond crystallography experiment at European XFEL. X-ray bursts strike crystallized samples resulting in diffraction patterns that can be reassembled into detailed images.
Credit: Shireen Dooling for the Biodesign Institute at ASU

Structure and functional binding epitopes of VISTA

V-domain Ig Suppressor of T-cell Activation (VISTA) is an immune checkpoint protein involved in the regulation of T cell activity. Checkpoint proteins are overexpressed by cancer cells or surrounding immune cells and prevent anti-tumor activity by co-opting natural regulation mechanisms to escape immune clearance. Compared to healthy tissues, VISTA is upregulated on tumor infiltrating leukocytes, including high expression on myeloid-derived suppressor cells (MDSCs). Through VISTA signaling, these inhibitory immune cells prevent effective antigen presentation and indirectly promote tumor growth. VISTA is implicated in a number of human cancers including skin (melanoma), prostate, colon, pancreatic, ovarian, endome­trial, and non-small cell lung. VISTA is a known member of the B7 protein family but the mechanism of action is still unclear as VISTA has been shown to function as both a ligand1,2 and a receptor3.  In the model of VISTA as a receptor, the proposed ligand of interaction is V-set and immunoglobulin domain containing 3 (VSIG3)4,5.

>Read more on the SSRL website

Image: Structure of human VISTA with extended C-C’ loop (blue), mapped VSTB/VSIG3 binding epitope (red), and disulfide bonds (yellow).

Discoveries map out CRISPR-Cas defence systems in bacteria

For the first time, researchers at the University of Copenhagen have mapped how bacterial cells trigger their defence against outside attacks. This could affect how diseases are fought in the future.

With the aid of highly advanced microscopes and synchrotron sources, researchers from the University of Copenhagen have gained critical insight into how bacteria function as defence mechanisms against attacks from other bacteria and viruses. The study, which has just been published in the renowned journal, Nature Communications, also describes how the defence systems can be activated on cue. This discovery can turn out to be an important cornerstone in fighting diseases in the future.

The researchers have shown how a cell attacked by a virus activates a molecule called COA (Cyclic Oligoadenylate), which in turn activates a so-called protein complex called CSX1 to eradicate the attacker.

>Read more on the MAX IV website

Image: Model of the CSX1 protein complex.

A citizen-science computer game for protein design

Using the computer game, “Foldit,” nonexpert citizen scientists designed new proteins whose structures, verified at the Advanced Light Source (ALS), were equivalent in quality to and more structurally diverse than computer-generated designs.

Proteins constitute the biomachinery—the cellular gears and levers—that make our bodies work. When this machinery is running smoothly, nutrients get absorbed, cells regenerate, and so on. When the machinery breaks down, the tools needed to fix the problem (i.e. drug molecules) are often proteins themselves.

Until recently, the pool of proteins available for such therapeutic purposes was limited to those found in nature. But natural proteins represent a small subset of all the possible ways to link 20 amino acids—the basic building blocks of all proteins—into chains hundreds, even thousands, of units long. On top of this, there are countless ways in which any given protein chain can fold—a key aspect of functionality.

In the last 20 years, “de novo” protein design (from scratch as opposed to starting with a known protein) has taken off, promising cheap and effective drugs with fewer side effects. But given the huge number of possibilities available, scientists are limited in their ability to fully explore this vast “protein space.”

>Read more on the Advanced Light Source website on Berkeley Lab

Image: The user interface of Foldit, a free online computer game developed to crowdsource problems in protein modeling. (a) The Foldit score: better models yield higher scores. (b) The design palette allows players to change the amino acids in the protein chain. (c) The “pull” tool allows players to manipulate the 3D structure of the model. (d) The “undo” graph tracks the score as a model is developed and allows players to backtrack. (e) Additional tool selections.

Preventing heart attacks

Scientists have taken an important step towards finding a potential cure for the disease that causes strokes and heart attacks in seniors and increases the mortality rate of diabetic and chronic kidney disease patients.
Researchers from the University of McGill and SickKids Toronto in collaboration with Universite de Montreal developed a simplified laboratory model that mimics the formation of mineral deposits that harden arteries and leads to these devastating conditions.
They used the Canadian Light Source (CLS) at the University of Saskatchewan to understand the type of minerals that formed and how they develop on the arteries.
“The goal in developing our lab model is that it would help us understand the mineralization process. We can then mimic what happens, and use it to test hypotheses on why the minerals are forming and also test some drugs to find something that can stop it,” said lead researcher Dr. Marta Cerruti.
Her six-member team is focused on the poorly understood process of how minerals form and grow on elastin, a protein on artery walls that provides the elasticity needed for blood flow to the heart, said Cerruti, an associate professor in Materials Engineering at McGill.
The hypothesis is that calcium phosphate-containing minerals form inside the walls of arteries and then calcify into a bone-like substance that narrows arteries and causes them to lose elasticity crucial for blood flow.

>Read more on the Canadian Light Source website

Image: Marta Cerruti (left) and Ophelie Gourgas in a laboratory using a Raman machine.

Mutated protein could become a non-hormonal contraceptive target

An international team of scientists from the Karolinska Institutet in Sweden and Nagoya University has explained how mutations in egg coat protein ZP1 cause infertility in women. The study suggests that ZP1 could be a promising candidate for future non-hormonal contraceptive efforts.
ZP1 is a glycoprotein involved in the fertilization of eggs by cross-linking egg coat filaments. Because studies in mice showed that lack of ZP1 reduces but does not abolish fertility, scientists believed that this molecule was also not crucial for fertility in humans. This new study, however, suggests that ZP1 may have a much more important role in human reproduction than previously thought. “The results were a big surprise because they suggested that mutations that truncate the human ZP1 protein cause female sterility by hindering its cross-linking function, rather than interfering with other egg coat proteins”, explains Luca Jovine, professor at the Karolinska Institutet and leader of the study.

>Read more on the ESRF website

Image: The mutation W83R of human ZP1 does not hinder its secretion but reduces its cross-linking (panel b), likely due to the fact that – as suggested by the structure of chicken ZP1 (panel a) – W83 (W72 in chicken ZP1) stacks between a sugar attached to ZP1 and the loop that makes the cross-link (“cd loop”). The part of the sugar chain that stacks against W83, which is a fucose residue, was only resolved in the structure of the fully glycosylated protein (violet) whose data came from ESRF ID23-1.

Potassium hunting on protein factories

Amazing insights into the location of elusive potassium ions on bacterial ribosomes

Groundbreaking research at the new long-wavelength macromolecular crystallography beamline (I23) at Diamond Light Source has for the first time demonstrated the location of potassium ions in bacterial ribosomes. Ribosomes are the protein factories of cells and although they are vital for life, little was known of the sites of metal ions that are crucial for their structure and function. The work recently published in Nature Communications showcases the fantastic applications of the I23 beamline and sheds light on the important role of potassium ions.

>Read more on the Diamond Light Source website

Image: (extract, full image here) 70S ribosome elongation complex (potassium atoms rendered as green spheres).

The interaction between two proteins involved in skin mechanical strength

A research team from the Centro de Investigación del Cáncer of the Universidad de Salamanca has obtained a detailed 3D image of the union between two hemidesmosomal proteins.

The structure of this complex has been unveiled using XALOC beamline techniques, at the ALBA Synchrotron. The results, published in “Structure”, provide insights to understand how these epithelial adhesion structures are formed. Researchers from Centro de Investigación del Cáncer – Instituto de Biología Molecular y Celular del Cáncer of Salamanca, from Centro Universitario de la Defensa in Zaragoza, and from the Netherlands Cancer Institute in Amsterdam have described how two essential proteins interact to each other in order to join epidermis and dermis together. This study reveals at atomic scale how the binding between two hemidesmosomal proteins called integrin α6β4 and BP230 takes place.
Epithelial tissues, such as epidermis, settle on fibrous sheets called basement membrane, formed by extracellular matrix proteins. The junction between epithelia and basement membrane happens through hemidesmosomes, multi-protein complexes located at the membrane of epithelial cells. Integrin α6β4 is an essential protein of the hemidesmosomes, which adheres to proteins of the basement membrane. Inside the cell cytoplasm, plectin and BP230 proteins bind to α6β4 and connect it to the intermediate filaments of the cytoskeleton. Genetic or autoimmune alterations that affect the hemidesmosomal proteins reduce skin resistance and cause diseases such as bullous pemphigoid and various types of epidermolysis bullosa.

>Read more on the ALBA website

Image: Structure of β4(WT)-BP230 complex.